RANK, an acronym for receptor activator of NF-κB, is a Type I transmembrane protein that is a member of the tumor necrosis factor (TNF) receptor superfamily and which when triggered activates the transcription factor NF-κB (Anderson et al., Nature 390:175-179 (1997); Anderson et al., U.S. Pat. No. 6,017,729). The human RANK (616 amino acids) has a signal peptide (28 amino acids), an N-terminal extracellular domain (184 amino acids), a short transmembrane domain (21 amino acids), and a large C-terminal cytoplasmic domain (383 amino acids), and mouse RANK is similarly arranged (Anderson et al., 1997). The extracellular domain of RANK contains four cysteine-rich pseudorepeats and two N-glycosylation sites, which features are characteristic of members of the TNF receptor superfamily. RANK has a 40% homology with CD40 (Anderson et al., 1997), and is expressed on T cells, dendritic cells, and osteoclasts (Anderson et al., 1997; Hofbauer et al., J Bone Min Res 15:2-12 (2000)).
The cytoplasmic domain of RANK associates intracellularly with several of the TNF receptor-associated factors (TRAFs; Baker and Reddy, Oncogene 12:1 (1996)) including TRAF1, TRAF2, TRAF3, TRAF5, and TRAF6 (Galibert et al., J Biol Chem 273:34120-27 (1998)). These TRAF binding sites are clustered in two distinct domains in the RANK cytoplasmic tail. The TRAFs are cytoplasmic proteins that often mediate signal transduction by members of the TNF receptor superfamily, and they are important in the regulation of, for example, immune and inflammatory responses. RANK mediates some or all of its biological activities through a cascade of events that involves the TRAF binding sites (see, for example, Galibert et al., 1998).
Triggering of RANK, such as by contact with membrane-bound or soluble RANK-L, results in the stimulation of RANK-mediated cellular responses. These cellular responses can include the activation of transcription factor NF-κB, a ubiquitous transcription factor that is extensively utilized in cells of the immune system, or the activation of Jun kinase (JNK; see, for example, Galibert et al., 1998). RANK activation in osteoclast progenitor cells induces the progenitors to differentiate into mature osteoclasts. This differentiation process is accompanied by the rearrangement of actin into “F-actin rings,” a specialized structure that is detectable by staining (Lakkakorpi, P. and Vaananen, J. Bone Min Res 6:817-826 (1991)). Elevated levels of c-src tyrosine kinase activity is also associated with RANK activation (Wong et al. Molecular Cell 4:1041-1049 (1999)).
RANK ligand (RANK-L) is a cell surface protein that binds with and activates RANK (Anderson et al., U.S. Pat. No. 6,017,729). This protein is also known as TRANCE, ODF or OPG ligand (Wong et al., 1999). RANK-L is a Type 2 transmembrane protein, and has an intracellular domain of about 50 amino acids or less, a transmembrane domain and an extracellular domain of about 240 to 250 amino acids. The extracellular domain of RANK-L contains a RANK-binding site. Similar to other members of the TNF family to which it belongs, RANK-L has a “spacer” region between the transmembrane domain and the receptor binding domain that is not necessary for receptor binding.
In bone, RANK-L stimulates osteoclast differentiation, enhances the activity of mature osteoclasts, and inhibits osteoclast apoptosis, thereby expanding the pool of activated osteoclasts (see, for example, Hsu et al., Proc Nat'l Acad Sci USA. 96:3540-45 (1999)). Osteoclasts are large, phagocytic, multinucleated cells which are formed from hematopoietic precursor cells in the bone marrow. Osteoclasts promote dissolution of the bone matrix and solubilization of bone salts, and are required for the proper development and growth of bones.
RANK knock-out mice are severely osteopetrotic and lack peripheral lymph nodes (Dougall et al., Genes Dev. 13:2412-24 (1999)). Modulation of RANK and RANK-L activity has been proposed as a means for treating a variety of disorders that involve osteopenia or osteopetrosis, including, for example, osteoporosis, Paget's disease, hypercalcemia, and so on (see, for example, WO 98/46751 and WO 99/58674).
RANK and its ligand play an integral role in the regulation of a wide range of biological systems, including the immune response, the inflammatory response, and bone remodeling through activation of osteoclasts. In view of the importance of RANK in the regulation of a wide range of biological systems, there is a need for methods for screening for molecules that antagonize or agonize RANK activity.